Regular Article

PHAGOCYTES, GRANULOCYTES, AND MYELOPOIESIS

A role for miR-155 in enabling tumor-infiltrating innate immune cells tomount effective antitumor responses in miceErika Zonari,1,2 Ferdinando Pucci,1,3 Massimo Saini,1,3 Roberta Mazzieri,1,3 Letterio S. Politi,4 Bernhard Gentner,1,3

and Luigi Naldini1,3

1San Raffaele Telethon Institute for Gene Therapy and Division of Regenerative Medicine, Gene Therapy and Stem Cells, San Raffaele Scientific Institute, Milan,

Italy; 2Universita degli Studi di Milano-Bicocca, Milan, Italy; 3Vita-Salute San Raffaele University, Milan, Italy; and 4Neuroradiology Division, Head and Neck

Department and Center of Excellence for High Field Magnetic Resonance Imaging, San Raffaele Hospital, Milan, Italy

Key Points

• miR-155 knockdown inmyeloid cells acceleratesspontaneous breast cancerdevelopment.

• miR-155 is required by TAMsfor deploying antitumoralactivity.

A productive immune response requires transient upregulation of the microRNA miR-155

in hematopoietic cellsmediating innate and adaptive immunity. In order to investigatemiR-

155 in the context of tumor-associated immune responses, we stably knocked down (KD)

miR-155 in themyeloid compartment of MMTV-PyMTmice, amousemodel of spontaneous

breast carcinogenesis that closely mimics tumor-host interactions seen in humans.

Notably, miR-155/KD significantly accelerated tumor growth by impairing classic activation

of tumor-associated macrophages (TAMs). This created an imbalance toward a protu-

moral microenvironment as evidenced by a lower proportion of CD11c1 TAMs, reduced

expression of activation markers, and the skewing of immune cells within the tumor

toward an macrophage type 2/T helper 2 response. This study highlights the importance

of tumor-infiltrating hematopoietic cells in constraining carcinogenesis and establishes an antitumoral function of a prototypical

oncomiR. (Blood. 2013;122(2):243-252)

Introduction

Innate and adaptive immune responses, modulated and potentiallysubverted by tumor cells, create an environment of chronic in-flammation that can turn a host defense mechanism into a promoterof tumor growth.1 Tumor progression has been associated with theexpansion of the myeloid compartment, and myeloid subsets com-posed of incompletely differentiated cells, often termed “myeloidderived suppressor cells,” have been found to possess protumoralproperties in experimental models.2 Within the tumor, myeloid cellsaccumulate mostly in the form of tumor-associated macrophages(TAMs), and their quantity correlates with worse prognosis in manytypes of tumors including those from Hodgkin lymphoma andbreast cancer.3,4 Instead, TAM infiltration can also be indicativeof an improved prognosis, for example, in colorectal cancer.5

TAMs are heterogeneous, with some subsets likely acting as bonafide ag-presenting cells (APCs) eliciting antitumor immunity, andothers fulfilling tissue remodeling functions and thus favoringtumor progression by supporting angiogenesis, invasion, andmetastasis.6,7 Our group has distinguished these functionallydifferent subsets by the expression of the tyrosine kinase receptorTIE2.6,8 Furthermore, we have identified 2 distinct geneexpression signatures in TIE21 TAMs (TEMs) and TIE22 TAMs,enabling their prospective isolation based on MRC1 and CD11cexpression, respectively.9 Whereas these data highlight the het-erogeneity of TAMs, little is known on the molecular mechanisms

underlying their activation and function within the tumormicroenvironment.

MicroRNAs (miRNAs) have emerged as global regulators ofgene expression programs.10 Recently, miR-511-3p, an miRNAencoded by the Mrc1 gene and thus associated with the macrophagesubset with tissue remodeling function, has been shown to providea negative feedback on protumoral genetic programs.11 On the otherhand, miRNAs can promote inflammation. miR-155 is upregulatedby immune stimuli such as antigens, Toll-like receptor ligands, andinflammatory cytokines in cells of the innate and adaptive immunesystem.12-17 miR-1552/2 mice have global immune defects charac-terized by decreased dendritic cell function and defective B- andT-cell responses.13-15 Specifically, the failure of miR-1552/2 miceto acquire protective immunity against Salmonella typhimuriuminfection was attributed to a failure of dendritic cells to efficientlyactivate T cells, probably due to a deficiency in antigen presentationand costimulation.13 The adaptive immune response in these micewas skewed toward a T helper 2 (Th2)–type immune responseassociated with the production of Th2 cytokines including interleukin(IL)-4, IL-5, and IL-10. These data strongly suggest that miR-155 isrequired for a T helper (Th1)–type response.

In this work, we investigated the function of miR-155 in TAMsusing a spontaneous breast cancer mouse model. We found thatmiR-155 is upregulated in CD11c1 pro-inflammatory TAMs.

Submitted August 23, 2012; accepted March 4, 2013. Prepublished online as

Blood First Edition paper, March 13, 2013; DOI 10.1182/blood-2012-08-

449306.

B.G. and L.N. share senior authorship.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge

payment. Therefore, and solely to indicate this fact, this article is hereby

marked “advertisement” in accordance with 18 USC section 1734.

© 2013 by The American Society of Hematology

BLOOD, 11 JULY 2013 x VOLUME 122, NUMBER 2 243

For personal use only.on January 5, 2015. by guest www.bloodjournal.orgFrom

Using a myeloid cell specific loss of function approach, weestablished that miR-155 is required for the activation of CD11c1

TAMs, and that this TAM subset, in turn, actively mediatesantitumor immunity, especially during the early stages of breastcarcinogenesis.

Material and methods

Lentiviral vectors

Third-generation lentiviral vectors (LVs) allowing stable knockdown oroverexpression of miR-155 were constructed and produced as previouslydescribed.18,19 See supplemental Methods for further details (on the BloodWeb site).

Cell lines

293T and RAW264.7 cells were maintained in Iscove modified Dulbeccomedium (Sigma-Aldrich) supplemented with 10% fetal bovine serum(Gibco) and a combination of penicillin-streptomycin and glutamine.

Mice

FVB and C57BL/6N wild-type mouse strains were purchased from CharlesRiver Laboratories, while FVB/PyMT mice were purchased from the NCI-Frederick Mouse Repositories. All mice were maintained in specific-pathogen-free conditions, and all procedures were performed accordingto protocols approved by the Animal Care and Use Committee ofthe Fondazione San Raffaele del Monte Tabor (IACUC 447, 455) andcommunicated to the Ministry of Health and local authorities according toItalian law.

HSPC transduction and transplantation

Bone marrow (BM) was harvested from the long bones of 6- to 8-week-olddonor mice (FVB or C57BL/6N females), and hematopoietic stem andprogenitor cells (HSPCs) were enriched by the depletion of lineage markerpositive cells using a mix of biotinylated monoclonal antibodies against Gr1,Ter-119, CD45R, CD3 (1:15), and CD11b (1:2000) (BD Pharmingen) and thesecondary reagents from the StemSep Mouse Progenitor Enrichment Kit(Stemcell Technologies). LV transduction was performed as previouslydescribed,18 and 106 cells were infused into the tail vein of lethally irradiated6-week-old female C57BL/6N or FVB mice, or 5.5-week-old FVB/MMTV-PyMTmice. Radiation doses were 1100 cGy for FVBmice and 1300 cGy forC57BL/6N mice. The majority of experiments were done using the FVBstrain, except those described in Figure 2C and supplemental Figures 2 and3A, which used C57BL/6N mice.

Magnetic resonance imaging (MRI)

MRI examinations of PyMT mice were performed on a 3.0 Tesla humanscanner (Achieva 3.0T; Philips), equipped with 80 mT/m gradients and a 40-mmvolumetric coil (Micro-Mouse 40). The mice were anesthetized with Avertinand fixed in a prone position on a dedicated temperature control apparatus. ATurbo Spin Echo T2 (repetition time = 2500; echo time = 80; turbo factor =9; field of view = 65 3 65 mm; zero-filled voxel size = 100 3 100 3 800mm) sequence was acquired. Tumor volumes were calculated after manualsegmentation of the lesion on the basis of signal intensity variation andenhancement characteristics by summing individual volumes (calculated aslesion area 3 slice thickness) of each slice.

Blood cell counts

Blood cell counts were determined using a Sysmex KX-21N apparatus. Foreach mouse, 10 mL of phosphate-buffered saline containing 45 mg/mLEDTA were added to 100 to 250 mL of peripheral blood.

Flow cytometry

For flow cytometry, we used a FACSCanto (BD Bioscience) platform. Seesupplemental Methods for further details.

Immunofluorescence staining and confocal microscopy

Tumors were cut into 12 to 16 mm cryostatic sections, as previouslydescribed.6 See supplemental Methods for further details.

Quantitative real-time polymerase chain reaction

Reactions were carried out in triplicate in an ABI Prism 7900HT Real-TimeSystem (Applied Biosystems). For quantification of miRNA expression,small RNAs were extracted using the miRNeasy Mini Kit (Qiagen), andmiRNA expression levels were determined by the Applied BiosystemsTaqMan MicroRNA Assay system. miRNA expression was normalized tomiR-16. Messenger RNA (mRNA) expression was quantified using eithermade-to-order TaqMan Gene Expression Assays (Applied Biosystems) ora custom-made TaqMan Low Density Array.9 See supplemental Methods forfurther details.

Statistical analysis

Unless otherwise noted, data are expressed as mean 6 standard error of themean (SEM). Statistical analyses were performed by Student t test, asindicated. Differences were considered statistically significant at P , .05.

Results

miR-155 expression in hematopoietic cells of normal and

tumor-bearing mice

In order to investigate the expression and biological activity of miR-155 in the hematopoietic cells of tumor-bearing mice, we used aspontaneous breast cancer model (PyMT) closely mimicking tumor-host interactions.20 Before tumor onset, these mice were reconstitutedwith BM cells transduced with a bidirectional miR-155 reportervector (BdLV.155T), which allows for the measurement of miR-155activity in hematopoietic cells of various organs at single cellresolution16,21 (Figure 1A). In tumor-infiltrating hematopoietic cells,we detected significant miR-155 activity in B cells and CD41 T cells,with the highest activity in regulatory T cells but not in CD81 T cellsor CD11b1 myeloid cells (Figure 1B). A similar picture was seen inthe spleen (Figure 1C). However, we noted low but significant miR-155 activity also in CD11b1 myeloid cells, a cell subset that isprogressively expanded with tumor growth. Because miR-155 isinduced by inflammatory stimuli,22 we measured miR-155 activity inthe splenocytes of BdLV.155T reconstituted wild-type mice thatwere or were not treated with lipopolysaccharide (LPS) (Figure 1D).LPS induced miR-155 activity, and this was most evident in CD11c1

APCs. A twofold to threefold induction of miR-155 activity couldalso be observed in vitro in primary BM-derived CD11c1 myeloidcells and in RAW264.7 cells after LPS stimulation, which corre-sponded to a sixfold induction of miR-155 expression as measured byquantitative polymerase chain reaction (qPCR) (Figure 1E; supple-mental Figure 1). Thus, miR-155 activity is constitutive in CD41

T cells and B cells and induced upon activation in CD11c1 APCs.Because the majority of TAMs infiltrating a late-stage tumor mightnot be in an activated stage, miR-155 activity might remainunnoticed when analyzing bulk CD11b1 cells. We thus sortedmyeloid subpopulations from the CD451 fraction of spontaneousmammary tumors taken from 13-week-old PyMTmice and measured

244 ZONARI et al BLOOD, 11 JULY 2013 x VOLUME 122, NUMBER 2

For personal use only.on January 5, 2015. by guest www.bloodjournal.orgFrom

Figure 1. miR-155 expression in hematopoietic cell subsets of normal and tumor-bearing mice. (A) Experimental scheme: The BM of 5.5-week-old PyMT mice was

reconstituted with wild-type FVB HSPCs transduced with miR-155 sensor (BdLV.155T) or Ctrl (BdLV.Ctrl) vectors. The chronology of breast cancer development and

progression is indicated. (B) Representative dot plots show reporter expression (y-axis: GFP, miRNA reporter; x-axis: NGFR, normalizer) in tumor-infiltrating CD41

lymphocytes (upper panel) or CD11b1 myeloid cells (lower panel) comparing BdLV.Ctrl (left) with BdLV.155T (right). Quantification of miR-155 activity in the indicated

tumor-infiltrating hematopoietic subpopulations was performed as described in Materials and methods. Shown is the mean “fold repression” of BdLV.155T with respect to

BdLV.Ctrl6 SEM, n 5 7 to 12 mice. (C) miR-155 activity in splenocytes recovered from 13-week-old PyMT mice is shown, along with the mean fold repression of BdLV.155T

with respect to BdLV.Ctrl6 SEM, n 5 6 to 10 mice. (D) The graph shows miR-155 activity in splenocytes of wild-type mice with (gray) or without (white) in vivo LPS treatment

(median 6 range, n 5 2). (E) Shown is the scheme of BM-derived dendritic cell (BMDC) differentiation protocol. miR-155 activity in BMDCs treated with LPS for 48 hours

(redundant, since we stated in materials and methods the we show the mean12 sem unless otherwise noted n5 3) is shown in the graph (bottom left). qPCR of the indicated

miRNAs in BMDCs that were or were not treated with LPS for 12 hours is shown in the graph (bottom right). The data show the relative abundance of each miRNA after

normalization to miR-16 (n 5 3). (F) Tumor-infiltrating hematopoietic cells were sorted from breast cancers of 13-week-old PyMT mice, as shown (top panels). miR-155

expression was measured in tumor-infiltrating macrophages (Mrc11 or CD11c1 subsets) and CD41 T lymphocytes by qPCR (bottom panel). Shown are the 22DCt values using

miR-16 as a normalizer (n 5 6 from 2 independent experiments). **P , .01; *** P , .001. eGFP, enhanced green fluorescent protein; NGFR, truncated low affinity nerve growth

factor receptor; PGK, phosphoglycerate kinase; SA, splice acceptor; SD, splice donor; WPRE, woodchuck hepatitis posttranscriptional regulatory element.

BLOOD, 11 JULY 2013 x VOLUME 122, NUMBER 2 miR-155 ENABLES ANTITUMOR RESPONSES 245

For personal use only.on January 5, 2015. by guest www.bloodjournal.orgFrom

the expression level of miR-155 by qPCR (Figure 1F).9 miR-155was expressed in CD11c1 TAMs at similar levels to those in CD41

T cells, but it was absent in the MRC11 protumoral macrophages,suggesting that miR-155 is activated in a subset of TAMs with pro-inflammatory and antigen-presenting functions.

miR-155/KD upregulates endogenous miR-155 targets and

attenuates AKT protein kinase signaling

In order to investigate whether miR-155 has a functional role ininnate immune cells of tumor-bearing mice, we developed a lentiviralsponge vector (supplemental Figure 2A)18 that allows stable knock-down of miR-155 (miR-155/KD) in myeloid cells, but it has littleactivity in T lymphocytes. To this aim, we exploited the spleen focusforming virus (SFFV) promoter, which drives transgene expressionto high levels in monocytes and dendritic cells, to intermediate levelsin granulocytes and B lymphocytes, and to very low levels inT lymphocytes, as shown in vivo after BM reconstitution by HSPCstransduced with green fluorescent protein (GFP) expressing SFFVLVs containing scrambled (Ctrl) or miR-155 sponge sequences(155/KD) (supplemental Figure 2B-C). The miR-155/KD vectorwas tested for its ability to knock down miR-155 in RAW264.7cells, which show basal miR-155 expression levels similar toinflammatory TAMs (see Figure 1F; supplemental Figure 1A). Wemeasured the expression levels of PU.1 protein, a previouslyreported miR-155 target,23 in RAW264.7 cells after transductionwith multiple copies of the miR-155/KD or Ctrl LV (Figure 2A).Both in steady state conditions and upon LPS treatment, PU.1 wasupregulated up to twofold times in miR-155/KD cells as measuredby western blot analysis (Figure 2A). Similarly, reverse transcrip-tion qPCR for Pu.1 and Ship1 mRNA, another well-establishedmiR-155 target,24 showed an ;1.5-fold upregulation upon miR-155/KD, while overexpression of miR-155 (miR-155/OE) usinga previously described LV design19 resulted in a 1.5 to twofolddownregulation (Figure 2B). Importantly, we showed a significantupregulation of Ship1 mRNA in splenic macrophages of micereconstituted with SFFV.155T/KD LV–transduced cells as opposedto SFFV.Ctrl LV–transduced cells after in vivo LPS challenge(Figure 2C). This result confirms the inhibition of miR-155regulation in myeloid cells in vivo by our vector. Whereas themajority of splenic macrophages expressed high levels of GFP,indicating strong expression of the sponge transcript and functionalinhibition of miR-155, only a minority of T cells expressed GFP tobarely detectable levels, consistently with low expression of thesponge promoter and functional activity of miR-155 in these cells.We then sorted and analyzed the subset of GFP-expressing T cellsand detected a minor modulation of the Ship1 target, which did notreach statistical significance. These data confirmed that, even in theminority of T cells in which sponge expression may have beenenhanced by integration site-specific effects or high vector copynumber, we did not achieve robust miR-155 knockdown. Thus, weconclude that our vector allows miR-155/KD in vivo specifically inmyeloid cells. We then investigated the impact of miR-155/KDon signaling pathways associated with Ship1, a major negativeregulator of growth factor receptor-mediated PI3K/AKT activationand myeloid cell survival (Figure 2D). Western blot analysis ofRAW264.7 cells indicated reduced levels of AKT phosphorylationat Ser473, a major activatory phosphorylation site, upon miR-155/KD(Figure 2E). This was accompanied by a reduction in phosphorylationof GSK3b at Ser9 and of S6 kinase at Ser235/236, 2 substrates ofAKT. Thus, miR-155/KD impairs AKT activation in macrophagesby increasing Ship1 levels. Next we tested whether the response

of macrophages to granulocyte monocyte colony stimulating factor(GM-CSF), a pro-inflammatory cytokine involved in the activationand maturation of APC signaling through the PI3K/AKT pathway,was altered upon miR-155/KD or OE. GM-CSF–induced AKT(Ser473) phosphorylation as assessed by phosphoflowwas reduced inmiR-155/KD cells and enhanced in miR-155/OE cells, and theseeffects were only evident when gating on the transduced cell popu-lation (Figure 2F). These data indicate that miR-155/KD alterssignaling pathways known to have a crucial function in inflam-matory macrophage activation.

Myeloid-specific miR-155 knockdown accelerates tumor growth

in a spontaneous breast cancer model

Next we addressed the question of whether miR-155 has a functionalrole in TAMs. To this aim, we transplanted PyMT mice with HSPCtransduced with multiple copies of the miR-155/KD vector (155/KDmice) or the Ctrl LV (Ctrl mice) (Figure 3A). At the time of transplant(5.5 weeks of age), PyMT mice showed hyperplasia of the breastepithelium with no detectable adenomas or carcinomas.25 Spontane-ous tumor development was monitored by MRI at 9 and 12 weeksof age (Figure 3B). Strikingly, miR-155/KD in BM-derived cellssignificantly increased tumor volume compared with the controlgroup, and this effect was already evident at the late adenoma stage(9-week time point). The early appearance of the effect furthersupports a myeloid cell–mediated phenotype, because T-cell re-constitution had not yet occurred 3.5 weeks after HSPC trans-plantation, in sharp contrast to myeloid lineage cells that show nearcomplete donor chimerism at 2 weeks (supplemental Figure 3A). Infact, ;50% of myeloid cells were miR-155/KD (estimated by GFPexpression) at the time of the first MRI, while ,0.7% of T cellsexpressed low levels of GFP (supplemental Figure 3B). Mice wereeuthanized between 12 and 13 weeks of age, and accelerated tumorgrowth in the miR-155/KD group was confirmed by the significantlyincreased weight of the surgically resected tumor masses (Figure 3C).Next we assessed the impact of miR-155/KD on hematopoiesis inPyMT mice. Blood cell counts did not show significant differencesearly after transplant, suggesting that miR-155/KD had no evidentimpact on hematopoietic reconstitution (Figure 3D). In addition,wild-type mice reconstituted with BM cells transduced with theSFFV.155T/KD vector and observed for 5 months showed no grossalterations in hematopoietic lineage counts in the peripheral blood,BM, spleen, and thymus, with the exception of a slight increasein B cells in the BM and spleen but not in the peripheral blood(supplemental Figure 2D-F). A similar phenotype was observed inmiR-1552/2mice,13 further confirming efficient miR-155 knockdownby our LV construct. Taken together, these data suggest that loss ofmiR-155 has no evident impact on hematopoietic reconstitution orsteady state myelo-, erythro-, and megakaryopoiesis. However, in12-week-old PyMT mice, tumor-induced anemia and leukocytosiswere significantly more severe in the miR-155/KD group comparedwith the scrambled Ctrl group (Figure 3E). Among the leukocytes,side scatter (SSC)low CD11b1monocytes showed the most significantincrease in miR-155/KD vs Ctrl mice (Figure 3F). These monocyteshave been reported to be protumoral and proangiogenic.26,27 Toinvestigate whether miR-155/KD in myeloid cells would alsoaccelerate the growth of established carcinomas, we set up anorthotopic breast cancer model. Tumors from 14-week-old PyMTmice (late carcinoma stage) were dissociated and orthotopicallyinjected into the mammary fat pad of FVB mice stablyreconstituted with HSPCs expressing miR-155/KD or Ctrl vector.Even though hematopoietic reconstitution with miR-155/KD cells

246 ZONARI et al BLOOD, 11 JULY 2013 x VOLUME 122, NUMBER 2

For personal use only.on January 5, 2015. by guest www.bloodjournal.orgFrom

Figure 2. miR-155/KD upregulates natural miR-155 targets and reduces AKT activation. (A-B) Deregulation of natural miR-155 targets in RAW264.7 cells upon stable

transduction with the indicated LVs is shown. (A) Western blot for PU.1 was performed on unstimulated and LPS-treated (1 mg/mL for 48 hours) cells. p84/Thoc1 and tubulin were

used as loading controls; densitometric quantification of the PU.1 band after normalization to p84 and calibration to the scrambled control is shown below the blots, together with

vector copy number in the tested cells. Blots are representative of 2 independent experiments. (B) qPCR for Pu.1 (left) and Ship1mRNA (right) was performed. Data shown are the

fold change to control, normalized to b2m (22ddCt); n5 6 to 19 independent experiments. Statistical analysis of the data was performed on 22DDCt values by unpaired Student t test.

(C) Shown are the results of qPCR for Ship1mRNA in GFP1 T cells and macrophages sorted from the spleens of wild-type mice reconstituted with Ctrl (white) or 155/KD (gray) LV-

transduced BMs 24 hours after treatment with 50 mg LPS intraperitoneally. n5 4 to 6 mice. (D) Shown is a simplified scheme of the PI3K/AKT signal transduction pathway. SHIP1,

a direct miR-155 target, is a major negative regulator of the PI3K/AKT axis. (E) Phosphorylation of PI3K downstream targets in macrophage RAW264.7 cells transduced with ScrT

Ctrl or miR-155/KD vectors (red); shown are western blots for pAKT (Ser473), pGSK3b (Ser9), and pS6 (Ser235/236). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was

used as a loading control. (F) The panels show GM-CSF induced activation of AKT in RAW264.7 cells upon miR-155/KD (GFP1 cells) or miR-155/OE (OFP1 cells). The pAKT

(S473) signal in vector-positive cells (middle histograms) and vector-negative cells (right histograms) is shown (FACS plots are representative). The colors represent the following:

red, miR-155/KD vector; blue, 155/OE vector; black, Ctrl vector-transduced RAW264.7 cells; and gray, miR-155/KD cells pretreated with the PI3K inhibitor Wortmannin (W) to set

the background signal. The MFI of each gated population is indicated. The column bar graph (right) shows pAKT Ser473 MFI in Raw264.7 cells transduced with the indicated

vectors. Statistical analysis was performed on n5 4 (155/KD) or n5 3 (155/OE) replicates using a paired Student t test against the corresponding Ctrl vector-transduced samples.

*P , .05; **P , .01. bT, bulged targets; MFI, mean flourescent intensity; pT, perfectly complementary targets; RTK, receptor tyrosine kinase; W, Wortmannin.

BLOOD, 11 JULY 2013 x VOLUME 122, NUMBER 2 miR-155 ENABLES ANTITUMOR RESPONSES 247

For personal use only.on January 5, 2015. by guest www.bloodjournal.orgFrom

was as high as that obtained in the PyMT mice, we observed nosignificant differences in tumor growth between the miR-155/KD andCtrl groups (Figure 3G). In summary, miR-155/KD accelerates tumor

development in a spontaneous mammary cancer model acting duringthe early stages of carcinogenesis by a mechanism involving myeloidcells.

Figure 3. miR-155 knockdown in myeloid cells accelerates tumor growth. (A) Experimental scheme: The BM of 5.5-week-old PyMT mice was reconstituted with

wild-type FVB HSPCs that were transduced with the miR-155/KD or ScrT Ctrl vectors. Mean vector copy numbers per cell 6 SEM are indicated. Chimeras are

referred to as PyMT Kd mice and PyMT Ctrl mice. (B) Mammary tumor growth was measured by MRI (mean volume 6 SEM of the 3 anterior mammary gland

pairs) at 9 and 12 weeks of age in PyMT Ctrl (n 5 9) and PyMT Kd (n 5 10) mice. (C) Tumor burden at the experimental end point (13 weeks) was determined by

weighing the mass of surgically resected tumor specimens from each mouse. Shown is the mean relative tumor weight in PyMT Ctrl mice (n 5 33) and PyMT Kd

mice (n 5 27). Within each of 4 independent experiments, tumor mass was normalized to the median tumor weight of the respective PyMT Ctrl group. (D) White

blood cell (WBC) count was measured in wild-type FVB mice (n 5 12), PyMT Ctrl (n 5 33), and PyMT Kd mice (n 5 39) at 9 weeks of age. (E) Complete blood cell

counts and hemoglobin levels were measured in wild-type FVB mice (n 5 12), PyMT Ctrl (n 5 30), and PyMT Kd mice (n 5 25) at 13 weeks of age. Results are from

4 independent experiments. (F) Differential WBC counts were obtained by multiplying the absolute WBC shown in (E) by the fraction of CD11b1side scatter (SSC)hi

cells (granulocytes, left panel), CD11b1SSClow cells (monocytes, middle panel), and CD191 cells (B lymphocytes, right panel) as measured by flow cytometry.

(G) The graph shows tumor growth in FVB mice transplanted with miR-155/KD or Ctrl vector–transduced HSPCs and orthotopically challenged with late-stage

PyMT mammary tumor cells 6 weeks after transplantation. Shown is the mean tumor weight 6 SEM; n 5 6 mice per group. HGB, hemoglobin; PLT, platelets; Wt,

wild type.

248 ZONARI et al BLOOD, 11 JULY 2013 x VOLUME 122, NUMBER 2

For personal use only.on January 5, 2015. by guest www.bloodjournal.orgFrom

miR-155 knockdown impairs the ability of myeloid cells to

mount a pro-inflammatory response

Tumor-infiltrating hematopoietic cells are important effectors of tumorrejection processes, especially during early-stage tumorigenesis,before tumor cells have acquired mechanisms of immune evasion.28

We quantified the composition of the inflammatory cell infiltrate in12-week-old PyMT tumors from either miR-155/KD or Ctrl mice. Thefrequency of infiltrating CD451 hematopoietic cells was similar intumor sections of miR-155/KD and control mice (data not shown), andflow cytometric analysis of dissociated tumors showed no differencesin the relative proportion of Gr11 cells, CD41, or CD81T and CD191

B lymphocytes (Figure 4A). Likewise, CD681 total macrophageswere equally represented in the 2 groups (Figure 4B). However, therewas a significant reduction in the expression of the activation markerionized binding adaptor molecule-1 (IBA-1) by innate immune cells inmiR-155/KD tumors (Figure 4B), and the myeloid infiltrate comprisedfewer CD11c1 cells as assayed by flow cytometry and qPCR(Figure 4C). These data suggest that miR-155/KD impairs activationof inflammatory macrophages in the tumor. We then performed geneexpression analysis on FACS-sorted, GFP1CD11b1F4/801 TAMsisolated from PyMT mammary tumors by using a custom-madeTaqMan Low Density Array carrying 95 genes including cytokines,chemokines, and genes involved in inflammation and immuneresponse (supplemental Table 1). Three genes were differentiallyexpressed between the miR-155/KD and Ctrl groups: interleukin(Il)-12b was significantly downregulated, while Il-4 and Il-13 were

significantly upregulated in the miR-155/KD myeloid infiltrate(Table 1). The same low density array was run on CD41 tumor-infiltrating lymphocytes (supplemental Table 2). Interestingly, Gm-csfand Il-13 were both significantly upregulated in T cells from the miR-155/KD group, whereas Il-6 and Ccr6 were significantly down-regulated (Table 2). These data support a skewing of the tumor immunemicroenvironment from a classic pro-inflammatory activation state toan alternatively activated phenotype comprising a Th2 profile.Collectively, these data highlight an impaired capacity of miR-155–deficient innate immune cells in establishing a pro-inflammatorymicroenvironment within the tumor, likely resulting in a reduction ofantitumor immune responses.

Discussion

Here we describe a key role of miR-155 in mediating the antitumorresponses of myeloid cells in vivo. By knocking down miR-155 inthe myeloid compartment of transgenic mice that spontaneouslydevelop breast cancer, we observed (1) an acceleration of tumorgrowth accompanied by a more severe tumor-induced anemia andmonocytosis; (2) a significant reduction in the proportion of CD11c1

inflammatory TAMs and in the expression of the macrophageactivation marker IBA-1; and (3) a skewing of the cytokine milieuof the tumor toward an M2/Th2 phenotype evident in the gene

Figure 4. miR-155 knockdown in myeloid cells

reduces TAMs with pro-inflammatory activity. (A)

The composition of hematopoietic infiltrate in PyMT

breast cancer was determined by flow cytometric

analysis of CD451 cells derived from dissociated

tumors. The proportion of Gr11 cells, CD41 or CD81

T lymphocytes, and CD191 B lymphocytes within the

total CD451 cells is shown (mean 6 SEM, n 5 9-10).

(B) Shown is representative immunofluorescence micros-

copy of tumor sections (top panels) from 13-week-old

PyMT Kd or PyMT Ctrl mice stained for the pan-

macrophage marker CD68 and IBA-1, a marker associ-

ated with macrophage activation. Original magnification

320. Shown is the quantification of the CD681 (bottom,

left panel) and IBA-11 (bottom, right panel) areas from

PyMT tumor sections relative to PyMT Ctrl (n 5 6 mice

from 2 independent experiments). (C) The graph (upper

panel) depicts the fraction of CD11c1 TAMs within the

total CD451 hematopoietic tumor infiltrate as measured by

flow cytometry. Shown are 2 independent experiments;

each point is a pool of 6 and 3 mice in experiment 1 and

experiment 2, respectively. The graph lower panel shows

the ratio of CD11c/Mrc1 transcript in the CD11b1F4/801

TAM population as measured by qPCR (n 5 3 pools of

3 mice each). Statistical analyses were performed by

Student t test. * P , .05.

BLOOD, 11 JULY 2013 x VOLUME 122, NUMBER 2 miR-155 ENABLES ANTITUMOR RESPONSES 249

For personal use only.on January 5, 2015. by guest www.bloodjournal.orgFrom

expression profile of both TAMs and CD41 TILs (tumor-infiltratinglymphocytes). Contrary to the prevailing view that myeloid cells favortumor growth and progression, our finding highlights an importantrole of hematopoietic cells in constraining tumor development andgrowth. By antagonizing an miRNA exclusively in hematopoieticcells, a comparably minor manipulation, we obtained a conspicuousacceleration of tumor growth in an already highly aggressivespontaneous breast cancer model.

Several lines of evidence suggest that the observed accelerationof tumor growth is mediated by miR-155 insufficiency in myeloidcells and, particularly, in APCs such as inflammatory macrophages/dendritic cells. These include the myeloid-biased expression of theknockdown vector, the occurrence of tumor acceleration beforethe onset of T-cell reconstitution following HSPC transplantation,and the changes in TAM phenotypes such as fewer CD11c1 cellsand the reduced expression of the activation marker IBA-1. Onthe contrary, we expect little/no influence of miR-155/KD on theprotumoral macrophage subsets because they do not expressrelevant levels of miR-155.

We investigated the molecular consequences of miR-155/KD ina macrophage cell line and in primary hematopoietic cells in vivoafter BM reconstitution. We found an upregulation of Ship1 and,consequently, a reduction in the activation of the PI3K/AKT pathwayat baseline and upon exposure to pro-inflammatory stimuli such asLPS and GM-CSF. SHIP1 is an inositol phosphatase and, togetherwith PTEN, the main negative regulator of PI3K/AKT signaling.29,30

It represents 1 of the main direct targets of miR-15531 and repressesthe production of pro-inflammatory cytokines, phagosome matura-tion, and macrophage survival.32 Consistently, AKT promotes theproduction of pro-inflammatory cytokines such as TNF-a, IL-6, andIL-12, while inhibiting the production of the anti-inflammatorycytokine IL-10.32 This might provide a mechanistic explanation forthe reduced occurrence of classically activated myeloid cells in miR-155/KD tumors and the M2/Th2-biased cytokine profile that weobserved in the tumor. In CD41 TILs, we found a significantupregulation of Il-13 andGm-csf, and we found a downregulation ofIl-6, Csf1r, and Pu.1 in the miR-155/KD group. IL-13 is a classicTh2 cytokine. In addition, downregulation of PU.1 might furtherreinforce Th2 functions because Pu.12/2 mice have been shown tohave a lower T-cell receptor activation threshold under Th2 cultureconditions.33 Instead, IL-6, which we found significantly down-regulated in the miR-155/KD group, is a classic pro-inflammatorycytokine.34 Macrophages and T cells interact closely, and it hasbeen suggested that they mutually polarize each other to a pro-inflammatory M1/Th1 phenotype on one end of the spectrum, or toan alternative M2/Th2 activation state on the other end.35 Thus, the

Th2 bias within the T-cell compartment might indirectly reflecta functional impairment of pro-inflammatory macrophages toinduce Th1 polarization, causing an imbalance toward pro-angiogenic, protumoral TAMs and a Th2 phenotype. Our geneexpression data, which show increased Il-4 and Il-13 and decreasedIl-12b levels in TAMs upon miR-155/KD, further support a shiftfrom M1 to M2 polarized macrophages. Recent reports suggestedthat miR-155 can directly modulate macrophage phenotypes.Arranz et al36 showed that a reduction in miR-155 directly inducesan M2 macrophage profile in mouse peritoneal macrophages byupregulating C/EBPb, a direct miR-155 target and a key regulatorof arginase 1 expression. Moreover, ectopic miR-155 expressioncan repolarize protumoral M2 TAMs toward an antitumoral M1phenotype,37 and augmenting miR-155 levels in tumor-associateddendritic cells by miRNA mimetics enhanced antitumor responsesin established ovarian cancers.38 These observations are fully inline with our data, which show impairment of classic macrophageactivation upon miR-155/KD. We do not exclude, however, thatmiR-155 insufficiency in other hematopoietic compartments mayhave similar protumoral effects, as recently proposed for T cells byHuffaker et al.39

It has been suggested that miR-155 positively regulates granu-locyte and monocyte development by acting on myeloid BMprogenitors,12 a compartment in which the SFFV-driven miR-155/KD vector is active.18 However, our in vivo results show an increasein the peripheral myeloid compartment (CD11b1 monocytes) uponmiR-155/KD. Thus, it is more likely that the expanded myeloidcompartment is a consequence of the increased tumor burden uponmyeloid-specific miR-155/KD rather than a direct effect of the miR-155/KD vector on the BM compartment. Indeed, wild-type miceshowed no alterations in blood cell counts when transplanted withmiR-155/KD LV–transduced hematopoietic progenitors. In supportof tumor-driven myeloid expansion, we noted increased GM-CSFexpression in CD41 TILs from the miR-155/KD group as comparedwith the control. GM-CSF is 1 of the principal myeloid growthfactors stimulating myelopoiesis and recruiting myeloid cells to thetumor.40 An additional mechanism of monocyte mobilization mightinvolve angiotensin receptor 1, which has been reported as a directmiR-155 target and is supposed to be upregulated upon miR-155/KD.41,42

Extensive data indicate that miR-155 functions as an oncomiR inseveral solid as well as hematologic tumors,43,44 and it is generallyconsidered a marker of poor prognosis.45,46 Constitutive miR-155expression in hematopoietic cells can result in the development of

Table 1. Low density gene expression TaqMan array performed ontumor-infiltrating macrophages from PyMT mice

Gene P value* Fold† dCt‡ SD

Il-12b ,.05 22.2 14.1 0.54

Il-4 ,.05 2.4 14.3 0.81

Il-13 ,.05 2.7 14.5 0.89

The tumor-infiltrating macrophages were sorted as GFP1CD451CD42CD11b1

F4/801cells and were from PyMT mice reconstituted with miR-155/KD– or Ctrl

LV–transduced HSPCs.

*Shown are differentially regulated genes with P value , .05. N = 3 replicates,

each representing a pool of 3 mice.

SD, standard deviation.

†The fold change of the respective transcripts in the miR-155/KD group with

respect to the control group is indicated (linear scale; “2” denotes “fold-less”

expression in the miR-155/KD samples).

‡Raw expression data were normalized to Actinb (dCT).

Table 2. Low density gene expression TaqMan array performed ontumor-infiltrating CD41 lymphocytes from PyMT mice

Gene P value* Fold† dCt‡ SD

Gm-csf ,.05 2.0 8.8 0.83

Il-13 ,.05 1.8 7.6 0.89

Ccr6 ,.05 21.8 12.8 0.91

Il-6 ,.05 22.4 14.2 0.91

Csf1r ,.05 22.4 12.0 1.15

Pu.1 ,.01 23.3 13.1 0.73

The tumor-infiltrating CD41 lymphocytes were sorted from PyMT mice

reconstituted with miR-155/KD– or Ctrl LV–transduced HSPCs. N 5 3 replicates,

each representing a pool of 3 mice.

SD, standard deviation.

*Shown are differentially regulated genes with P value , .05.

†The fold change of the respective transcripts in the miR-155/KD group with

respect to the control group is indicated (linear scale; “2” denotes fold-less

expression in the miR-155/KD samples).

‡Raw expression data were normalized to Actinb (dCT).

250 ZONARI et al BLOOD, 11 JULY 2013 x VOLUME 122, NUMBER 2

For personal use only.on January 5, 2015. by guest www.bloodjournal.orgFrom

hematologic malignancies such as lymphoma and myeloproliferativedisease.12,45,47 These evidences have motivated the development ofanti–miR-155 drugs as potential anticancer therapeutics.43,48-50 Ourdata indicate that the oncogenic activity of miR-155 is cell contextdependent, and thus suggest some caution before applyinganti–miR-155 treatments to cancer patients. In this regard, itshould be kept in mind that miR-155 has a physiological role inorchestrating a productive immune response. Previous work hashighlighted the importance of miR-155 in acute inflammatoryinsults.13,14,22 Our data expand the role of miR-155 to myeloidcell–mediated antitumoral responses, because blocking its physio-logical upregulation in these cells accelerated tumor development ina spontaneous breast cancer model. Thus, a wave of miR-155activation in differentiated hematopoietic cells boosts immunity,and only failure to downregulate miR-155 may support oncogen-esis, suggesting that miR-155 is, first of all, an “immunomiR” ratherthan an oncomiR. It remains to be determined whether the potentialbenefits of miR-155 inhibition (ie, reduction in cell autonomoustumor cell growth in selected cancers with miR-155 overexpression)outweigh immunosuppression, which can, paradoxically, favortumor growth.

In summary, we here describe an antitumoral role of miR-155 asan integral effector of immunosurveillance, thus limiting early stepsof breast cancer development. This work highlights the contextspecificity and complexity of miRNA regulation, calling for a celltype specific approach when targeting miRNA function, which takesinto consideration the heterogeneity of tumors composed of bothcancer cells and a collection of nonmalignant host cells. Moreover,our findings support the concept that TAMs make up distinguishablesubsets within the tumor, which mediate different and sometimesopposing functions in tumor development and growth.

Acknowledgments

The authors thank Alice Giustacchini, Maura Rossetti, AndreaAnnoni, and Nada Sonda for help with some experiments; AlessioPalini for cell sorting; Clelia Di Serio for the original design of thestatistical analysis for the low density array; Lucia Sergi Sergi forvector production; and Giulia Schira, Anna Ranghetti, and DavideMoi for technical help with some experiments. The authors also thankMichele De Palma and Vincenzo Bronte for helpful discussions.

This work was supported by grants from the Italian Associationfor Cancer Research and the European Union (ERC AdvancedGrant 249845, TARGETINGGENETHERAPY) (L.N.). E.Z. wassupported by a fellowship from the Italian Foundation for CancerResearch.

Authorship

Contribution: E.Z. designed and performed research, analyzed data,and drafted the manuscript; F.P. analyzed gene expression data; M.S.performed western blot and phosphoflow analyses; R.M. discussedthe data; L.S.P. performed MRI; and L.N. and B.G. conceived andcoordinated the research, analyzed data, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competingfinancial interests.

Correspondence: Luigi Naldini, San Raffaele Telethon Institutefor Gene Therapy, Via Olgettina, 58, 20132 Milan, Italy; e-mail:naldini.luigi@hsr.it.

References

1. Hanahan D, Coussens LM. Accessories to the crime:functions of cells recruited to the tumormicroenvironment. Cancer Cell. 2012;21(3):309-322.

2. Gabrilovich DI, Nagaraj S. Myeloid-derivedsuppressor cells as regulators of the immunesystem. Nat Rev Immunol. 2009;9(3):162-174.

3. Leek RD, Lewis CE, Whitehouse R, Greenall M,Clarke J, Harris AL. Association of macrophageinfiltration with angiogenesis and prognosis ininvasive breast carcinoma. Cancer Res. 1996;56(20):4625-4629.

4. Steidl C, Lee T, Shah SP, et al. Tumor-associatedmacrophages and survival in classic Hodgkin’slymphoma. N Engl J Med. 2010;362(10):875-885.

5. Sconocchia G, Zlobec I, Lugli A, et al. Tumorinfiltration by FcgRIII (CD16)1 myeloid cells isassociated with improved survival in patients withcolorectal carcinoma. Int J Cancer. 2011;128(11):2663-2672.

6. De Palma M, Venneri MA, Galli R, Sergi Sergi L,Politi LS, Sampaolesi M, Naldini L. Tie2 identifiesa hematopoietic lineage of proangiogenicmonocytes required for tumor vessel formationand a mesenchymal population of pericyteprogenitors. Cancer Cell. 2005;8(3):211-226.

7. Qian BZ, Pollard JW. Macrophage diversityenhances tumor progression and metastasis.Cell. 2010;141(1):39-51.

8. Venneri MA, De Palma M, Ponzoni M, et al.Identification of proangiogenic TIE2-expressingmonocytes (TEMs) in human peripheral blood andcancer. Blood. 2007;109(12):5276-5285.

9. Pucci F, Venneri MA, Biziato D, et al. Adistinguishing gene signature shared bytumor-infiltrating Tie2-expressing monocytes,

blood “resident” monocytes, and embryonicmacrophages suggests common functions anddevelopmental relationships. Blood. 2009;114(4):901-914.

10. Bartel DP. MicroRNAs: target recognition andregulatory functions. Cell. 2009;136(2):215-233.

11. Squadrito ML, Pucci F, Magri L, et al. miR-511-3pmodulates genetic programs of tumor-associatedmacrophages. Cell Rep. 2012;1(2):141-154.

12. O’Connell RM, Rao DS, Chaudhuri AA, et al.Sustained expression of microRNA-155 inhematopoietic stem cells causes amyeloproliferative disorder. J Exp Med. 2008;205(3):585-594.

13. Rodriguez A, Vigorito E, Clare S, et al.Requirement of bic/microRNA-155 for normalimmune function. Science. 2007;316(5824):608-611.

14. Thai TH, Calado DP, Casola S, et al. Regulationof the germinal center response by microRNA-155. Science. 2007;316(5824):604-608.

15. Lu LF, Thai TH, Calado DP, et al. Foxp3-dependent microRNA155 confers competitivefitness to regulatory T cells by targeting SOCS1protein. Immunity. 2009;30(1):80-91.

16. Brown BD, Gentner B, Cantore A, et al.Endogenous microRNA can be broadly exploitedto regulate transgene expression according totissue, lineage and differentiation state. NatBiotechnol. 2007;25(12):1457-1467.

17. Tili E, Michaille JJ, Cimino A, et al. Modulationof miR-155 and miR-125b levels followinglipopolysaccharide/TNF-alpha stimulation andtheir possible roles in regulating the response

to endotoxin shock. J Immunol. 2007;179(8):5082-5089.

18. Gentner B, Schira G, Giustacchini A, AmendolaM, Brown BD, Ponzoni M, Naldini L. Stableknockdown of microRNA in vivo by lentiviralvectors. Nat Methods. 2009;6(1):63-66.

19. Amendola M, Passerini L, Pucci F, Gentner B,Bacchetta R, Naldini L. Regulated and multiplemiRNA and siRNA delivery into primary cells bya lentiviral platform. Mol Ther. 2009;17(6):1039-1052.

20. Lin EY, Jones JG, Li P, Zhu L, Whitney KD, MullerWJ, Pollard JW. Progression to malignancy in thepolyoma middle T oncoprotein mouse breastcancer model provides a reliable model for humandiseases. Am J Pathol. 2003;163(5):2113-2126.

21. Gentner B, Visigalli I, Hiramatsu H, et al.Identification of hematopoietic stem cell-specificmiRNAs enables gene therapy of globoid cellleukodystrophy. Sci Transl Med. 2010;2(58):58ra84.

22. O’Connell RM, Taganov KD, Boldin MP, Cheng G,Baltimore D. MicroRNA-155 is induced during themacrophage inflammatory response. Proc NatlAcad Sci USA. 2007;104(5):1604-1609.

23. Vigorito E, Perks KL, Abreu-Goodger C, et al.microRNA-155 regulates the generation ofimmunoglobulin class-switched plasma cells.Immunity. 2007;27(6):847-859.

24. Androulidaki A, Iliopoulos D, Arranz A, et al. Thekinase Akt1 controls macrophage response tolipopolysaccharide by regulating microRNAs.Immunity. 2009;31(2):220-231.

25. De Palma M, Mazzieri R, Politi LS, et al. Tumor-targeted interferon-alpha delivery by Tie2-

BLOOD, 11 JULY 2013 x VOLUME 122, NUMBER 2 miR-155 ENABLES ANTITUMOR RESPONSES 251

For personal use only.on January 5, 2015. by guest www.bloodjournal.orgFrom

expressing monocytes inhibits tumor growth andmetastasis. Cancer Cell. 2008;14(4):299-311.

26. Sica A, Bronte V. Altered macrophagedifferentiation and immune dysfunction in tumordevelopment. J Clin Invest. 2007;117(5):1155-1166.

27. Yang L, DeBusk LM, Fukuda K, et al. Expansionof myeloid immune suppressor Gr1CD11b1 cellsin tumor-bearing host directly promotes tumorangiogenesis. Cancer Cell. 2004;6(4):409-421.

28. Schreiber RD, Old LJ, Smyth MJ. Cancerimmunoediting: integrating immunity’s roles incancer suppression and promotion. Science.2011;331(6024):1565-1570.

29. Baran CP, Tridandapani S, Helgason CD,Humphries RK, Krystal G, Marsh CB. The inositol59-phosphatase SHIP-1 and the Src kinase Lynnegatively regulate macrophage colony-stimulating factor-induced Akt activity. J BiolChem. 2003;278(40):38628-38636.

30. Mondal S, Subramanian KK, Sakai J, Bajrami B,Luo HR. Phosphoinositide lipid phosphataseSHIP1 and PTEN coordinate to regulate cellmigration and adhesion.Mol Biol Cell. 2012;23(7):1219-1230.

31. O’Connell RM, Chaudhuri AA, Rao DS, BaltimoreD. Inositol phosphatase SHIP1 is a primary targetof miR-155. Proc Natl Acad Sci USA. 2009;106(17):7113-7118.

32. Rajaram MV, Butchar JP, Parsa KV, Cremer TJ,Amer A, Schlesinger LS, Tridandapani S. Akt andSHIP modulate Francisella escape from thephagosome and induction of the Fas-mediateddeath pathway. PLoS ONE. 2009;4(11):e7919.

33. Chang HC, Han L, Jabeen R, Carotta S, Nutt SL,Kaplan MHPU. PU.1 regulates TCR expression

by modulating GATA-3 activity. J Immunol. 2009;183(8):4887-4894.

34. Mihara M, Hashizume M, Yoshida H, Suzuki M,Shiina M. IL-6/IL-6 receptor system and its role inphysiological and pathological conditions. Clin Sci(Lond). 2012;122(4):143-159.

35. Sica A, Mantovani A. Macrophage plasticity andpolarization: in vivo veritas. J Clin Invest. 2012;122(3):787-795.

36. Arranz A, Doxaki C, Vergadi E, et al. Akt1 andAkt2 protein kinases differentially contribute tomacrophage polarization. Proc Natl Acad SciUSA. 2012;109(24):9517-9522.

37. Cai X, Yin Y, Li N, et al. Re-polarization of tumor-associated macrophages to pro-inflammatory M1macrophages by microRNA-155. J Mol Cell Biol.2012;4(5):341-343.

38. Cubillos-Ruiz JR, Baird JR, Tesone AJ, et al.Reprogramming tumor-associated dendritic cellsin vivo using miRNA mimetics triggers protectiveimmunity against ovarian cancer. Cancer Res.2012;72(7):1683-1693.

39. Huffaker TB, Hu R, Runtsch MC, et al. Epistasisbetween microRNAs 155 and 146a during T cell-mediated antitumor immunity. Cell Rep. 2012;2(6):1697-1709.

40. Hamilton JA. Colony-stimulating factors ininflammation and autoimmunity. Nat RevImmunol. 2008;8(7):533-544.

41. Sethupathy P, Borel C, Gagnebin M, et al. HumanmicroRNA-155 on chromosome 21 differentiallyinteracts with its polymorphic target in the AGTR139 untranslated region: a mechanism for functionalsingle-nucleotide polymorphisms related tophenotypes. Am J Hum Genet. 2007;81(2):405-413.

42. Swirski FK, Nahrendorf M, Etzrodt M, et al.Identification of splenic reservoir monocytes andtheir deployment to inflammatory sites. Science.2009;325(5940):612-616.

43. Iorio MV, Croce CM. MicroRNA dysregulation incancer: diagnostics, monitoring and therapeutics.A comprehensive review. EMBO Mol Med. 2012;4(3):143-159.

44. Volinia S, Calin GA, Liu CG, et al. A microRNAexpression signature of human solid tumorsdefines cancer gene targets. Proc Natl Acad SciUSA. 2006;103(7):2257-2261.

45. Yanaihara N, Caplen N, Bowman E, et al. UniquemicroRNA molecular profiles in lung cancerdiagnosis and prognosis. Cancer Cell. 2006;9(3):189-198.

46. Chen J, Wang BC, Tang JH. Clinical significanceof microRNA-155 expression in human breastcancer. J Surg Oncol. 2012;106(3):260-266.

47. Lee DW, Futami M, Carroll M, et al. Loss of SHIP-1 protein expression in high-risk myelodysplasticsyndromes is associated with miR-210 and miR-155. Oncogene. 2012;31(37):4085-4094.

48. Gambari R, Fabbri E, Borgatti M, et al. TargetingmicroRNAs involved in human diseases: a novelapproach for modification of gene expression anddrug development. Biochem Pharmacol. 2011;82(10):1416-1429.

49. Lovat F, Valeri N, Croce CM. MicroRNAs in thepathogenesis of cancer. Semin Oncol. 2011;38(6):724-733.

50. Kong YW, Ferland-McCollough D, Jackson TJ,Bushell M. microRNAs in cancer management.Lancet Oncol. 2012;13(6):e249-e258.

252 ZONARI et al BLOOD, 11 JULY 2013 x VOLUME 122, NUMBER 2

For personal use only.on January 5, 2015. by guest www.bloodjournal.orgFrom

online March 13, 2013 originally publisheddoi:10.1182/blood-2012-08-449306

2013 122: 243-252  

Gentner and Luigi NaldiniErika Zonari, Ferdinando Pucci, Massimo Saini, Roberta Mazzieri, Letterio S. Politi, Bernhard mount effective antitumor responses in miceA role for miR-155 in enabling tumor-infiltrating innate immune cells to 

http://www.bloodjournal.org/content/122/2/243.full.htmlUpdated information and services can be found at:

(491 articles)Phagocytes, Granulocytes, and Myelopoiesis    (5253 articles)Immunobiology   

Articles on similar topics can be found in the following Blood collections

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://www.bloodjournal.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

http://www.bloodjournal.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

  Copyright 2011 by The American Society of Hematology; all rights reserved.of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society

For personal use only.on January 5, 2015. by guest www.bloodjournal.orgFrom